Rapid Induction of Neural Differentiation in Human Umbilical Cord Matrix Mesenchymal Stem Cells by cAMP-elevating Agents.

Human umbilical cord matrix (hUCM) is considered as a promising source of mesenchymal stem cells (MSCs) due to several advantages over other tissues. The potential of neural differentiation of hUCM-MSCs is of great interest in the context of treating neurodegenerative diseases. In recent years, considerable efforts have been made to establish in vitro conditions for improving the differentiation of hUCM-MSCs toward neuronal cells. In the present study, we evaluated the neural differentiation potential of hUCM-MSCs in the presence of cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). hUCM-MSCs were isolated from fetal umbilical cord and characterized by ﬂow cytometry analysis for mesenchymal specific markers. Mesodermal differentiation potential was assessed through selective media with lineage-specific induction factors. For assessment of neural differentiation, cells were cultured in the presence of cAMP-elevating agents for 8 and 24 h. The neuronal differentiated MSCs were characterized for neuronal specific markers by immunocytochemistry and western blotting. Isolated hUCM-MSCs were found positive for mesenchymal markers (CD73, CD90, and CD105) while negative for hematopoietic markers (CD34 and CD45) .Following neural induction, most cells represented neural-like cells morphology. Neural markers including β-tubulin III (Tuj-1), neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2) and nestin were expressed in treated cells with respect to control group. The astrocyte specific marker, glial fibrillary acidic protein (GFAP) was also shown by immunofluorescence in treated cells. (These findings demonstrate that hUCM-MSCs have the ability to rapidly differentiate into neural cell types of neuron-like cells and astrocytes by cAMP-elevating agents without the presence of growth factors.

, chondrocytes (2) and adipocytes (3). Due to their easy accessibility, straightforward procedures of isolation and immunoregulatory property, MSCs are good candidates to be used in cell therapy (4). It has been reported that MSCs can also transdifferentiate into other cell types derived from germ layers including cardiomyocytes (5), hepatocytes (6) and neurocytes (7). In particular, several studies have shown the differentiation potential of MSCs into neurallike cells (8,9). The use of MSCs for treatment of neurodegenerative diseases has become of interest. The potential application of MSCs in neurodegenerative diseases is based on their transdifferentiation capability into neural cells (10) in addition to their neuroprotective and immunoregulatory properties (11). Therefore, it is not surprising that the clinical applications of these stem cells for Parkinson's disease (12), multiple sclerosis (13), Alzheimer's disease (14), amyotrophic lateral sclerosis (ALS) (15) and Huntington's disease will increase in the coming years (16).
The mesenchymal stem cells can be classified into two categories: MSCs derived from adult tissues such as bone marrow (17), adipose tissue (18), endometrial polyps (19) and MSCs derived from fetal/perinatal tissues such as placenta (20), amniotic membrane (21), umbilical cord blood and matrix (22). The common source of human MSCs is bone marrow (BM-MSCs); however, BM-MSCs comprise very small fraction (0.001% to 0.01%) of the total population of nucleated cells in the marrow and their proliferative capacity and differentiation potential decrease with age (23). Therefore, finding alternative sources of MSCs would be beneficial for both therapeutic and research purposes. Umbilical cord matrix (also known as the Wharton's jelly) is emerging as a promising source of MSCs obtained by non-invasive methods. In contrast to BM-MSCs, umbilical cord matrix-MSCs (UCM-MSCs) have faster proliferation and greater ex vivo expansion capacity that might be due to the expression of telomerase by these cells (24).
In addition, UCM-MSCs are more primitive than mesenchymal stem cells derived from other tissues and have the ability to remain undifferentiated for at least 10 passages in vitro (25).
Interestingly, transplantation of UCM-MSCs is not associated with teratoma formation despite the primitive features of these cells (26).
Thus, the umbilical cord matrix represents a pro-  After 3 days of culture, the non-adherent cells were removed by changing the medium. The cells were passaged and expanded when they had grown to 80% to 90% confluence. All experiments were carried out by MSCs between passages 1 to 3.  binding, the membrane was placed in a film cassette, exposed to chemiluminescence (ECL, Amersham Bioscience, USA) reagent and X-ray film (Thermo Scientific) for various time periods. Consistent with the previous reports (1,29), these cells were uniformly positive for mesenchymal markers (CD73, CD105, and CD90) and were found negative for hematopoietic stem cell markers (CD34, CD45) ( Figure 1).

In vitro differentiation potential of hUCM-MSCs
To determine whether UCM-MSCs are able to differentiate into mesodermal cell types, the cells were cultured in selective media. The differentiation capacity of these cells into adipocytes, osteoblasts and chondrocytes were verified by specific staining (Figure 2).    Figure 4A).

Expression of neural markers using western blot analysis
It is believed that cAMP-dependent protein kinase A (PKA) is the main effector for cAMP signal in eukaryotic cells (30). cAMP-mediated PKA activation induces cAMP-response elementbinding protein (CREB) phosphorylation (31,32).
Thus, to indicate the role of cAMP levels in neural differentiation of hUCM-MSCs, a phospho-specific antibody was used to examine the phosphorylation of CREB at Ser-133. As presented in Figure 5A,

Discussion
Stem cell-based therapies are promising approaches for the treatment of neurodegenerative diseases (12,33). Recently, MSC has created major breakthroughs in the field of regenerative medicine.
hUCM is considered as a promising source of